Carrier system for specific artery wall gene delivery

ABSTRACT

An artery wall binding peptide (AWBP) based on the artery wall cell-binding domain of apolipoprotein B-100 was conjugated to a cationic backbone configured for forming a complex with a nucleic acid to produce a composition that enhances gene transfer to artery wall cells. An illustrative cationic backbone is poly(ethylene glycol)-grafted-poly(L-lysine) (PEG-g-PLL). Methods of making and using the composition for gene transfer are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/247,320, filed Nov. 10, 2000.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. government support under Grant No.HL-65477 awarded by the National Institutes of Health. The U.S.government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

This invention relates to gene delivery. More particularly, thisinvention relates to compositions of matter, methods of use thereof, andmethods of making thereof for delivering genes.

Gene therapy provides significantly important opportunities for treatingvarious kinds of life-threatening and gene-related disease by producingbiologically active agents or stopping abnormal functions of the cells,such as genetic failure or uncontrollable proliferation of cells. Actualapplication of genes to human therapy is limited by several problems,including their instability in body fluids, non-specificity to thedesired cells, degradation by nucleases, and low transfectionefficiency. Gene delivery systems have been investigated in attempts toenhance gene expression and reduce cytotoxicity. S.-O. Han et al.,Development of Biomaterials for Gene Therapy, 2 Mol. Ther. 302-317(2000).

Among the various gene delivery systems, viral vectors, M. A. Rosenfieldet al., Adenovirus-mediated Transfer of a Recombinant A1-antitrypsineGene to the Lung Epithelium In Vivo, 252 Science 431-434 (1991); H. M.Temin, Safety Considerations in Somatic Gene Therapy of Human Diseasewith Retrovirus Vectors, 1 Hum. Gene Ther. 111-123 (1990), liposomalcarriers, X. Gao & L. Huang, Cationic Liposome-mediated Gene Delivery, 2Gene Ther. 710-722 (1995); A. R. Thierry et al., Systemic Gene Delivery:Biodistribution and Long-term Expression of a Transgene in Mice, 92Proc. Nat'l Acad. Sci. USA 9742-9746 (1995); J. H. Senior et al.,Interaction of Positively-charged Liposomes with Blood: Implications forTheir Application In Vivo, 1070 Biochim. Biophys. Acta 173-179 (1991),and cationic polymers, Y.-B. Lim et al., Biodegradable Polyester, Poly[α-(4-amino-butyl)-L-glycolic acid], as a Non-toxic Gene Carrier, 17Pharm. Res. 811-816 (2000); P. Lemieux et al., Block and GraftCopolymers and NanoGel Copolymer Networks for DNA Delivery into Cell, 8J. Drug. Target. 91-105 (2000), S.-O. Han et al., Water SolubleLipopolymer for Gene Delivery, 12 Bioconjug. Chem. 337-345 (2001), havebeen widely investigated in gene therapy areas. Although retroviruses,adenoviruses, and adeno-associated viruses have shown highertransfection efficiency in vitro, the application of viral vectors tothe human body is also limited by safety problems such as the immuneresponse against transfection systems, oncogenic effects, and thepotential ability of endogenous virus recombination. These problems havestimulated the development of non-viral gene delivery. As non-viralvectors, liposomes and cationic polymers have been extensivelyinvestigated for a decade due to the advantages of safety and relativelylow cost. Although higher transfection efficiency has been reported byliposomal gene carriers in vitro, A. R. Thierry et al., supra, someliposomal gene carriers are unstable in aqueous solution and aggregatein blood. J. H. Senior et al., supra. Cationic polymers includingpoly(L-lysine) (“PLL”) and polyethyleneimine (“PEI”) were able tocondense plasmid DNA and protect it from enzymatic degradation, whichresulted in enhancement of transfection efficiency. However, drawback,such as biocompatibility in the body, still remain before such polymerscan be used for gene delivery. To overcome the biocompatibility problem,non-toxic biodegradable polymeric gene carriers have been developed aspromising gene delivery materials. Y.-B. Lim et al., supra. However, thebiodistribution of the polymer/pDNA complexes following the injection ofcomplexes into the body is still unknown. For the enhanced delivery ofgenes to specific cells, polymeric gene carriers have been modified withspecific cell targeting moieties such as galactose, M. Nishikawa et al.,Hepatocyte-targeted In Vivo Gene Expression by Intravenous Injection ofPlasmid DNA Complexed with Synthetic Multi-functional Gene DeliverySystem, 7 Gene Ther. 548-555 (2000), transferrin, E. Wagner et al.,Influenza Virus Hemaglutinin HA-2 N-terminal Fusogenic Peptides AugmentGene Transfer by Transferrin-polylysine-DNA complexes: Toward aSynthetic Virus-like Gene-transfer Vehicle, 89 Proc. Nat'l Acad. Sci.USA 7934-7938 (1992), and antibody, W. Suh et al., Anti-JL1 AntibodyConjugated Poly(L-lysine) for Targeted Gene Delivery to Leukemia TCells, 72 J. Control. Release 171-178 (2001).

Recently, a series of methoxy poly(ethyleneglycol)-grafted-poly(L-lysine (PEG-g-PLL) gene carriers was synthesizedfor reducing cytotoxicity, increasing solubility in aqueous solution,and enhancing the transfection efficiency resulting from long-termexpression compared to PLL in a human carcinoma cell line. Y. H. Choi etal., Polyethylene Glycol-grafted Poly-L-lysine as Polymeric GeneCarrier, 54 J. Control. Release 39-48 (1998). A lactose group was alsocoupled to the end of PEG-g-PLL for specific targeting to hepatomacells. Y. H. Choi et al., Lactose-poly(ethylene glycol)-graftedPoly-L-lysine as Hepatoma Cell-targeted Gene Carrier, 9 Bioconjug. Chem.708-718 (1998); Y. H. Choi et al., Characterization of a Targeted GeneCarrier, Lactose-Polyethylene Glycol-grafted Poly-L-lysine, and itsComplex with Plasmid DNA, 10 Hum. Gene Ther. 2657-2665 (1999).Transfection efficiency of such Lac-PEG-g-PLL/pDNA complexes wasincreased several-fold higher than that of PLL/DNA complexes in Hep G2cells. A7R5 and NIH 3T3 cell lines do not have lactose receptors ontheir surfaces; consequently, the transfection efficiency ofLac-PEG-g-PLL/pDNA complexes was much lower than in the Hep G2 cells.

It was well known that low-density lipoprotein (LDL) can be taken up bydifferent types of cells (vascular endothelial cells, vascular smoothmuscle cells, hepatocytes, and macrophages) via receptor-mediatedendocytosis. In previous reports, J. S. Kim et al., In Vitro GeneExpression on Smooth Muscle Cells Using a Terplex Delivery System, 47 J.Control. Release 51-59 (1997); J. S. Kim et al., Terplex DNA DeliverySystem as a Gene Carrier, 15 Pharm. Res. 116-121 (1998), a terplex-DNAgene delivery system comprising plasmid DNA, low density lipoprotein(LDL), and hydrophobized poly(L-lysine) (H-PLL) enhanced gene transfervia the LDL receptor-mediated endocytosis pathway. The transfectionefficiency of the terplex-DNA system was 2-5 times higher than that ofLipofectin™/pDNA in A7R5 murine smooth muscle cells. Lipofectin™ reagentis a 1:1 (w/w) liposome formulation of the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA),and dioleoyl phosphotidylethanolamine (DOPE) in membrane filtered water.This system also showed significantly higher transfection efficienciesin vitro in artery wall cells, L. Yu et al., Terplex DNA Gene CarrierSystem Targeting Artery Wall Cells, 72 J. Control. Release 179-189(2001), and in vivo in myocardium cells, D. G. Affleck et al.,Augmentation of Myocardia Transfection Using Terplex DNA: a Novel GeneDelivery System, 8 Gene Ther. 349-353 (2001).

Gene delivery systems containing a specific cell-targeting moiety havethe advantage in the efficient delivery to the desired cells or organs.Although PLL has been described as an efficient gene carrier, U. K.Laemmli, Characterization of DNA Condensates Induced by Poly(ethyleneoxide) and Polylysine, 72 Proc. Nat'l Acad. Sci. USA 4288-4299 (1975),as an alternative to liposomes or viral vectors, PLL/pDNA complexesdisplayed some limitations such as the precipitation of PLL/pDNAcomplexes in high concentration and low biocompatibility in the humanbody. Y. H. Choi et al., 54 J. Control. Release 39-48 (1998),investigated PEGylated-PLL/pDNA complexes to overcome these limitationsof PLL by conjugation of PEG to PLL. Although PEGylated-PLL was shown tobe a biocompatible material in tissues, efficient transfection tospecific cells still remained a problem to overcome.

In view of the foregoing, it will be appreciated that providing acomposition for matter for specific gene delivery to artery wall cellswould be a significant advancement in the art.

BRIEF SUMMARY OF THE INVENTION

An illustrative composition of matter according to the present inventioncomprises an artery wall binding peptide covalently coupled to apharmaceutically acceptable cationic backbone, wherein the cationicbackbone is configured for complexing with a nucleic acid. Inillustrative embodiments of this invention, the artery wall bindingpeptide is SEQ ID NO:2 or a biologically functional equivalent thereof.In another illustrative embodiment of this invention, the artery wallbinding peptide is present in a molar ratio to the cationic backbone ofgreater than 1:1. In still another illustrative embodiment of thisinvention, the artery wall binding peptide is present in a molar ratioto the cationic backbone of at least 2:1. The cationic backbone cancomprise, for example, a cationic polymer, a cationic lipid, or amixture thereof. Illustrative cationic polymers according the inventioninclude poly(L-lysine), poly(ethyleneimine), polyamidoamine dendrimer,poly[α-(4-aminobutyl)-L-glycolic acid], chitosan,poly(2-dimethylamino)ethyl methacrylate, poly(ethyleneglycol)-grafted-poly(L-lysine), and the like.

Another illustrative composition of matter according to the presentinvention has the formula:(AWBP)_(n)-PEG-g-PLLwherein AWBP is an artery wall binding peptide, n is an integer of atleast 1, and PEG-g-PLL is poly(ethylene glycol-grafted-poly(L-lysine).In other illustrative embodiments of this invention n is about 4 and/orAWBP is SEQ ID NO:2.

Still another illustrative composition of matter according to thepresent invention comprises an artery wall binding peptide (SEQ ID NO:2)covalently coupled to poly(ethylene glycol)-grafted-poly(L-lysine). Inanother illustrative embodiments of this invention the artery wallbinding peptide (SEQ ID NO:2) is covalently coupled to poly(ethyleneglycol)-grafted-poly(L-lysine) in a molar ratio of about 4:1.

Yet another illustrative composition of matter according to the presentinvention comprises an artery wall binding peptide covalently coupled topoly(ethylene glycol)-grafted-poly(L-lysine). In other illustrativeembodiments of this invention the artery wall binding peptide iscovalently coupled to poly(ethylene glycol)-grafted-poly(L-lysine) in amolar ratio of about 4:1 and/or the artery wall binding peptide is SEQID NO:2.

An illustrative pharmaceutical composition according to the presentinvention comprises a mixture of:

(a) an effective amount of a composition comprising an artery wallbinding peptide covalently coupled to a pharmaceutically acceptablecationic backbone, wherein the cationic backbone is configured forcomplexing with a nucleic acid; and

(b) a pharmaceutically acceptable carrier.

Another illustrative pharmaceutical composition according to the presentinvention comprises a mixture of:

(a) an effective amount of a conjugate represented by the formula:(AWBP)_(n)-PEG-g-PLLwherein AWBP is an artery wall binding peptide, n is an integer of atleast 1, and PEG-g-PLL is poly(ethylene glycol)-grafted-poly(L-lysine);and

(b) a pharmaceutically acceptable carrier.

Still another illustrative pharmaceutical composition according to thepresent invention comprises a mixture of:

(a) an effective amount of a composition comprising artery wall bindingpeptide covalently coupled to poly(ethyleneglycol)-grafted-poly(L-lysine); and

(b) a pharmaceutically acceptable carrier.

An illustrative method of making a composition having the formula:(AWBP)_(n)-PEG-g-PLLwherein AWBP is an artery wall binding peptide, n is an integer of atleast 1, and PEG-g-PLL is poly(ethylene glycol)-grafted-poly(L-lysine),comprises:

(a) conjugating poly(ethylene glycol) to poly(L-lysine) to result inpoly(ethylene glycol)-grafted-poly(L-lysine); and

(b) conjugating artery wall binding peptide to the poly(ethyleneglycol)-grafted-poly(L-lysine) to result in (AWBP)_(n)-PEG-g-PLL.

An illustrative method for delivering a nucleic acid to a cell bearing areceptor that binds an artery wall binding peptide comprises:

(a) mixing the nucleic acid with a composition of matter comprising anartery wall binding peptide covalently coupled to a cationic backbone,wherein the cationic backbone is configured for complexing with thenucleic acid, to form a complex;

(b) causing the complex to contact the cell such that the receptor bindsthe artery wall binding peptide, thereby delivering the nucleic acid tothe cell.

Another illustrative embodiment of a method for delivering a nucleicacid to a cell bearing a receptor that binds an artery wall bindingpeptide comprises:

(a) mixing the nucleic acid with a composition of matter comprising anartery wall binding peptide covalently coupled to poly(ethyleneglycol)-grafted-poly(L-lysine) to result in a complex comprising anucleic acid portion, a poly(ethylene glycol)-grafted-poly(L-lysine)portion, and an artery wall binding peptide portion; and

(b) causing the complex to contact the cell such that the receptor bindsthe artery wall binding peptide portion, thereby delivering the nucleicacid to the cell.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows an illustrative scheme for synthesis of AWBP-PEG-g-PLLcomprising artery wall binding peptide (AWBP, SEQ ID NO:2 shown insingle-letter code) conjugated to PEG-g-PLL.

FIG. 2A shows the results of matrix-assisted laser desorption-time offlight (MALDI-TOF) mass spectrometry of AWBP.

FIG. 2B shows a ¹H-NMR spectrum of AWBP.

FIG. 2C shows a ¹H-NMR spectrum of AWBP-PEG-g-PLL.

FIG. 3A shows a gel band shift assay of AWBP-PEG-g-PLL/pDNA complexes:lane 1, 300 ng of 1 kbp DNA step ladder molecular mass marker; lane 2,360 ng of plasmid DNA; lanes 3-10, charge ratio of polymer/plasmidDNA=0.1, 0.2, 0.5, 1, 2, 3, 5, and 10, respectively.

FIG. 3B shows a DNase protection assay of AWBP-PEG-g-PLL/pDNA complexes:lane 1, 100 bp DNA step ladder; lane 2, plasmid DNA; lanes 3-9,incubation times of 0, 5, 10, 15, 30, 60, 120 minutes, respectively.

FIG. 4 shows particle size distributions of AWBP-PEG-g-PLL/pDNAcomplexes measured by zeta potentiometer.

FIG. 5 shows surface morphology of an AWBP-PEG-g-PLL/pDNA complex (2/1,+/−) measured by atomic force microscopy (AFM).

FIGS. 6A and 6B show AWBP-PEG-g-PLL mediated gene transfer (open bars)to bovine aorta endothelial cells (A) and smooth muscle cells (B); PLL(shaded bars) and PEG-g-PLL (dark bars) were used as negative controlgene carriers.

FIGS. 7A and 7B show inhibition of AWBP-PEG-g-PLL mediated gene transfer(open bars) to bovine aorta endothelial cells (A) and smooth musclecells (B) with free AWBP; PLL (shaded bars) and PEG-g-PLL (dark bars)were used as negative control gene carriers.

DETAILED DESCRIPTION

Before the present carrier system for specific artery wall gene deliveryis disclosed and described, it is to be understood that this inventionis not limited to the particular configurations, process steps, andmaterials disclosed herein as such configurations, process steps, andmaterials may vary somewhat. It is also to be understood that theterminology employed herein is used for the purpose of describingparticular embodiments only and is not intended to be limiting since thescope of the present invention will be limited only by the appendedclaims and equivalents thereof.

The publications and other reference materials referred to herein todescribe the background of the invention and to provide additionaldetail regarding its practice are hereby incorporated by reference. Thereferences discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to a pharmaceutical composition comprising “a pharmaceuticallyacceptable carrier” includes a mixture of two or more of such carriers,reference to “an artery wall binding protein” includes reference to oneor more of such artery wall binding proteins, and reference to “aplasmid” includes reference to a mixture of two or more of suchplasmids.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

As used herein, “comprising,” “including,” “containing,” “characterizedby,” and grammatical equivalents thereof are inclusive or open-endedterms that do not exclude additional, unrecited elements or methodsteps. “Comprising” is to be interpreted as including the morerestrictive terms “consisting of” and “consisting essentially of.”

As used herein, “consisting of” and grammatical equivalents thereofexclude any element, step, or ingredient not specified in the claim.

As used herein, “consisting essentially of” and grammatical equivalentsthereof limit the scope of a claim to the specified materials or stepsand those that do not materially affect the basic and novelcharacteristic or characteristics of the claimed invention.

As used herein, “single-letter code” and similar terms refer tosingle-letter designations for the 20 amino acid residues found inpeptides and proteins, as follows: A—alanine, C—cysteine, D—asparticacid, E—glutamic acid, F—phenylalanine, G—glycine, H—histidine,I—isoleucine, K—lysine, L—leucine, M—methionine, N—asparagine,P—proline, Q—glutamine, R—arginine, S—serine, T—threonine, V—valine,W—tryptophan, and Y—tyrosine.

As used herein, “pDNA” means plasmid DNA.

As used herein, “cationic backbone” means a cationic molecule, complex,or conjugate, or the like, configured for forming a complex with anucleic acid. Illustrative cationic backbones include cationic polymersand cationic lipids. Illustrative cationic polymers that can be usedwithin the scope of the present invention include poly(L-lysine) (PLL),poly(ethyleneimine) (PEI), polyamidoamine dendrimer,poly[α-(4-aminobutyl)-L-glycolic acid] (PAGA), chitosan,poly(2-dimethylamino)ethyl methacrylate (pDMAEMA), PEG-g-PLL, and thelike. An illustrative cationic lipid isN-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA).

As used herein, “artery wall binding peptide” or “AWBP” mean a peptideconfigured for binding to a receptor that binds the artery wallcell-binding domain of apo B-100. According to the present invention, aligand comprising such an artery wall binding peptide is coupled to acationic backbone, such as PEG-g-PLL, so that upon endocytosis of theAWBP ligand any nucleic acid complexed to the cationic backbone moietyis also internalized by the cells.

Illustrative artery wall binding peptides include the peptide having theamino acid sequence identified as SEQ ID NO:2 and biologicallyfunctional equivalents thereof. Such functional equivalents retainfunctionality in binding the receptor and eliciting receptor-mediatedendocytosis although they may be truncations, deletion variants, orsubstitution variants of SEQ ID NO:2 or include additional amino acidresidues attached thereto.

As mentioned above, changes may be made in the structure of the arterywall binding peptide while maintaining the desirable receptor-bindingcharacteristics. For example, certain amino acid residues may besubstituted for other amino acid residues in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies or binding sitesof ligands such as an artery wall binding peptide. Since it is theinteractive capacity and nature of a protein that defines that protein'sbiological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence and nevertheless obtaina protein with like properties. It is thus contemplated that variouschanges may be made in the sequence of an artery wall binding peptidewithout appreciable loss of its biological utility or activity.

It is also well understood by the skilled artisan that inherent in thedefinition of a biologically functional equivalent protein or peptide isthe concept that there is a limit to the number of changes that may bemade within a defined portion of the molecule and still result in amolecule with an acceptable level of equivalent biological activity. Itis also well understood that where certain residues are shown to beparticularly important to the biological or structural properties of aprotein or peptide, e.g. residues in active sites, such residues may notgenerally be exchanged.

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chains relative to, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. An analysisof the size, shape, and type of the amino acid side-chains reveals, forexample, that arginine, lysine, and histidine are all positively chargedresidues; that alanine, glycine, and serine are all a similar size; andthat phenylalanine, tryptophan, and tyrosine all have a generallysimilar shape. Therefore, based upon these considerations, the followingconservative substitution groups or biologically functional equivalentshave been defined: (a) Cys; (b) Phe, Trp, Tyr; (c) Gln, Glu, Asn, Asp;(d) His, Lys, Arg; (e) Ala, Gly, Pro, Ser, Thr; and (f) Met, Ile, Leu,Val. M. Dayhoff et al., Atlas of Protein Sequence and Structure (Nat'lBiomed. Res. Found., Washington, D.C., 1978).

To effect more quantitative changes, the hydropathic index of aminoacids may be considered. Each amino acid has been assigned a hydropathicindex on the basis of its hydrophobicity and charge characteristics,which are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine(+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan(−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate(−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine(−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art. J. Kyte & R. Doolittle, A Simple Method for Displaying theHydropathic Character of a Protein, 157 J. Mol. Biol. 105-132 (1982). Itis known that certain amino acids may be substituted for other aminoacids having a similar hydropathic index or score and still retain asimilar biological activity. In making changes based on the hydropathicindex, the substitution of amino acids whose hydropathic indices arewithin ±2 is preferred, within ±1 is particularly preferred, and within±0.5 is even more particularly preferred.

It is also understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine(−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4).

In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, within ±1 is particularly preferred, and within ±0.5 is evenmore particularly preferred.

Therefore, biologically functional equivalents of AWBP can be discoveredwithout undue experimentation by a person of ordinary skill in the artaccording to the guidance and principles disclosed herein.

As used herein, a “pharmaceutically acceptable” component is one that issuitable for use with humans and/or animals without undue adverse sideeffects (such as toxicity, irritation, and allergic response)commensurate with a reasonable benefit/risk ratio. Illustratively, a“pharmaceutically acceptable” component includes one that is approved bya regulatory agency of the U.S. or other national government or a stategovernment or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the composition is administered. Such pharmaceutical carrierscan be sterile liquids, such as water or aqueous saline solutions andaqueous dextrose and glycerol solutions.

As used herein, “effective amount” means an amount of a composition orpharmacologically active agent that is nontoxic but sufficient toprovide the desired local or systemic effect and performance at areasonable benefit/risk ratio attending any medical treatment.

As used herein, “administering” and similar terms mean delivering thecomposition to the individual being treated such that the composition iscapable of being circulated systemically to the parts of the body wherethe AWBP portion of the composition can bind its receptor, e.g. arterywalls. Thus, the composition is preferably administered to theindividual by systemic administration, typically by subcutaneous,intramuscular, or intravenous administration, or intraperitonealadministration. Injectables for such use can be prepared in conventionalforms, either as a liquid solution or suspension or in a solid formsuitable for preparation as a solution or suspension in a liquid priorto injection, or as an emulsion. Suitable excipients include, forexample, water, saline, dextrose, glycerol, ethanol, and the like; andif desired, minor amounts of auxiliary substances such as wetting oremulsifying agents, buffers, and the like can be added.

Apolipoprotein B-100 (apo B-100), a major protein component of LDL,contains many receptor-binding domains, such as LDL receptor-bindingdomain, artery wall cell-binding domain, and heparin-binding domain. Ithas been demonstrated that a synthetic peptide containing amino acidresidues 1000-1016 of apo B-100(Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Phe-Val-Thr-Gln-Ala-Glu-Gly-Ala-Lys;SEQ ID NO:1) is the arterial binding domain of apo B-100. I. L. Shih etal., Focal Accumulation of an Apolipoprotein B-based SyntheticOligopeptide in the Healing Rabbit Arterial Wall, 87 Proc. Nat'l Acad.Sci. USA 1436-1440 (1990). The focal accumulation of ¹²⁵I-labeledapoB-based synthetic peptide at the healing edges of regeneratingendothelial islands in balloon-catheter deendothelialized rabbit aortasuggested that this arterial wall-binding peptide could mediateaccumulation of LDLs in arterial lesions. I. L. Shih et al., supra.

In the present invention, a synthetic peptide based on the arterialbinding domain of apo B-100 was selected as a ligand for binding thecompositions of the present invention to artery wall cells. A cysteineresidue was added to the amino-terminus of the peptide to facilitateconjugation of the peptide to a cationic backbone. SEQ ID NO:2 shows theamino acid sequence of such a peptide.

In the present invention, a conjugate comprising AWBP covalently coupledto a cationic backbone was designed as a specific cell-targeting genedelivery system to artery wall cells. The artery wall is an attractivetargeting tissue for gene therapy because it is distributed through allorgans of the body. Although vascular gene transfer has beendemonstrated with various techniques, a gene delivery system forarterial wall targeting has not previously been developed for theefficient treatment of cardiovascular diseases such as atherosclerosisand restenosis.

The gene delivery compositions of the present invention are made bycovalently binding the artery wall binding peptide to the cationicbackbone according to methods well known in the art. For example, aminogroups on the peptide can react with acid chlorides of carboxylic acidsto yield amide linkages or acid chlorides of sulfonic acids to yieldsulfonamide linkages. By way of further example, amino groups can reactwith alkyl halides to yield alkylated amine linkages. Further,carboxylic acid groups on peptides can react with OH groups on acationic backbone to form ester linkages, with amino groups to formamide linkages, and with SH groups to form thioester linkages. Stillfurther, many crosslinking compounds are known in the art and arecommercially available for crosslinking a peptide to, for example, apolymer or a lipid. Example 1 illustrates a vinylsulfone crosslinker forconjugating the SH group of cysteine to poly(ethylene glycol).

The compositions of the present invention are used by first mixing witha nucleic acid to be delivered to result in a complex. The positivecharges on the composition interact with the negative charges on thenucleic acid to form the complex. The complex is then administered tothe individual to be treated by gene therapy. The complex is prepared ina conventional form by mixture with a carrier, as described above. Themixture is then is administered to the individual by systemicadministration, typically by subcutaneous, intramuscular, or intravenousadministration, or intraperitoneal administration.

The size of the gene carrier and carrier/pDNA complex has beenconsidered an important factor for enhancing the transfection efficiencysince the particle size of the polymer/pDNA complex was reported toaffect the transfection efficiency. J. Y. Cherng et al., Effect of Sizeand Serum Proteins on Transfection Efficiency ofPoly((2-dimethylamino)ethyl methacrylate)-plasmid Nanoparticles, 13Pharm. Res. 1038-1042 (1996). C. X. Song et al., Arterial Uptake ofBiodegradable Nanoparticles for Intravascular Local Drug Delivery:Results with an Acute Dog Model, 54 J. Control. Release 201-211 (1998),reported a potentially useful particle size of about 70-160 nm for localintraluminal therapy of restenosis. Since drug carriers with a smallerparticle size have resulted in higher arterial uptake compared tocarriers with larger size, the size of the complexes was expected to bea dominating factor in the arterial wall lesions of the rapid blood flowwhich could wash out most of the drugs or therapeutic chemical agentsfrom the arterial wall lesions within 20-30 minutes. Illustratively, theAWBP-PEG-g-PLL/pDNA complex according to the present invention has asize of about 100 nm (see Example 3), which is estimated as being anacceptable size for particles targeted for arterial wall lesions.

In gene expression studies designed to illustrate the operability andefficiency of compositions of the present invention (Example 4),transfection efficiencies of AWBP-PEG-g-PLL/pDNA complexes to bovineaorta endothelial cells and smooth muscle cells were 150-180 timeshigher than those of control carriers, PLL and PEG-g-PLL. In atransfection inhibition study (Example 4), luciferase activities ofAWBP-PEG-g-PLL/pDNA complexes in both cells were significantly decreasedwith increase of free AWBP concentrations. However, the luciferaseactivities of control systems, such as PLL/pDNA complexes andPEG-g-PLL/pDNA complexes, were not significantly changed with theincrease of free AWBP concentration. These results indicated that genetransfer of AWBP-PEG-g-PLL/pDNA complexes clearly proceeded by areceptor-mediated endocytosis pathway. Thus, it was clearly demonstratedthat AWBP-PEG-g-PLL could function as a targeted gene delivery carrierto arterial wall cells via receptor-mediated endocytosis.

Progression of atherosclerotic lesions is marked by accumulation ofaltering layers of smooth muscle cells and endothelial cells. Therefore,the higher transfection efficiency of AWBP-PEG-g-PLL/pDNA complexes inthese cells might be useful to evaluate the potential ability of todeliver a gene to the artery wall cells. The selective interactionsbetween vascular endothelial cells and circulated complexes could beapplied for a potential therapeutic approach. The rational design of thechemical structure of polymeric gene carriers such as AWBP-PEG-g-PLLwith higher gene transfection efficiency and tissue specific genedelivery in vitro may become a very promising non-viral gene deliverysystem for cardiovascular gene therapy. Also, it is expected that thedevelopment of AWBP-PEG-g-PLL will be a turning point in the clinicaltherapy of artery related diseases such as athersclerosis andrestenosis.

EXAMPLE 1

Synthesis of AWBP-PEG-g-PLL. Fifty milligrams of PLL hydrobromide (120repeating units, M_(r) 25,000, Sigma Chemical Co., St. Louis, Mo.) wasdissolved in 1.0 ml of PBS (0.01 M Na₂HPO₄, 0.15 M NaCl, pH 6.5) and thesolution was stirred for 40 min. at room temperature. Next, 27.2 mg ofN-hydroxysuccinimide polyethylene glycol vinylsulfone (NHS-PEG-VS, M_(r)3400, Shearwater Polymers, Huntsville, Ala.) in 1.0 ml of dimethylsulfoxide (DMSO, Aldrich, St. Louis, Mo.) was slowly added to the PLLsolution and the reaction mixture was stirred for 3 hours at roomtemperature. After dialysis against distilled water in a dialysis tubing(Spectrum, Houston, Tex.) with a molecular weight cut-off of 15,000 for1 day, the product was obtained by lyophilization to yield 60.0 mg ofVS-PEG-g-PLL (77.6 wt. %).

Four milligrams of artery wall binding peptide (AWBP, M_(r) 2008, SEQ IDNO:2; Genemed Synthesis, South San Francisco, Calif.) in 0.5 ml DMSO wasadded dropwise into a solution of 28.1 mg VS-PEG-g-PLL in 2.0 ml PBS (pH7.0), and the reaction mixture was stirred for 6 hours at roomtemperature. The mixture was dialyzed for 4 days as described above andthen lyophilized. The amount of final product was 20.0 mg ofAWBP-PEG-g-PLL (62.2 wt. %). The final product was analyzed by 400 MHz¹H NMR (Varian, Palo Alto, Calif.) and then stored at −20° C. beforeuse.

FIG. 1 illustrates the synthesis of a conjugate of artery wall bindingpeptide (AWBP; SEQ ID NO:2) to PEG-g-PLL, which conjugate is termedAWBP-PEG-g-PLL. The synthetic scheme comprises two reactions, first thesynthesis of an activated PEG-g-PLL having a vinylsulfone group attachedto the PEG portion of PEG-g-PLL, and then conjugation of AWBP to thevinylsulfone group to result in AWBP-PEG-g-PLL. Briefly, in the firststep the N-hydroxysuccinimide (NHS) group of NHS-PEG-VS was conjugatedto the amino group of PLL. The structure of the product and theconjugation reaction were analyzed by ¹H-NMR as shown in FIGS. 2A-C. Thecontent of PEG was estimated from the ¹H-NMR analysis by the relativeareas of alkyl groups in NHS-PEG-VS (—CH₂CH₂—, s, 3.21-3.77 ppm) andthose of the side chains of PLL (—CH₂CH₂CH₂—, m, 1.05-1.90 ppm). In asecond reaction, AWBP was conjugated to the end of the vinylsulfonegroup of VS-PEG-g-PLL. ¹H-NMR analysis determined that 4 mol of AWBPwere reacted with one mole of VS-PEG-g-PLL by the comparison of peaks at7.3 ppm (aromatic group from phenylalanine) and 0.5-1.5 ppm (lysine peakfrom PLL). In addition, the specific proton peak (11.85-12.61 ppm) (FIG.2B) of the thiol group on AWBP totally disappeared in the spectraobtained of AWBP-PEG-g-PLL (arrow in FIG. 2C), which indicated that thethiol groups of the peptide were completely conjugated to thevinylsulfone group of VS-PEG-g-PLL.

EXAMPLE 2

Gel band shift and DNase protection assay. A plasmid encoding fireflyluciferase driven by the cytomegalovirus (CMV) promoter was constructedby inserting the luciferase gene into the mammalian gene expressionplasmid pMNK at the MluI and KpnI restriction sites (Promega, Madison,Wis.). The plasmid DNA was transformed into Escherichia coli DH5α andamplified in terrific broth at 37° C. overnight with vigorous shaking at225 rpm. The amplified plasmid DNA was purified using a Qiagen Maxiplasmid purification kit. The purity and concentration of the obtainedplasmid DNA in Tris-EDTA (TE) buffer were determined by ultraviolet (UV)absorbance at 260 nm. The optical density ratios at 260 to 280 nm of theplasmid DNA were in the range of 1.7-1.8. The absence of generearrangement during cloning and propagation of the plasmid DNA wasconfirmed by restriction digest using SalI and EcoRI (BoehringerMannheim GmbH, Germany) and 1% agarose gel electrophoresis.

AWBP-PEG-g-PLL/pDNA complexes were prepared at various charge ratiosranging from 0.1/1 to 20/1 (+/−) in HEPES-buffered saline (15 mM HEPES,150 mM NaCl, pH 7.3) (HBS) and incubated for 20 minutes at roomtemperature. Afterwards, the samples were fractionated byelectrophoresis through a 0.8% agarose gel at 100 V for 40 minutes andstained with ethidium bromide (0.5 μg/ml) for 45 minutes. DNA was thenvisualized with a UV illuminator.

AWBP-PEG-g-PLL/pDNA complexes were prepared at the charge ratios of 2/1(+/−) and incubated in the presence of 10 times excess of DNase I. At 0,5, 10, 15, 20, 60, and 120 minutes after incubation, 50 μl of the samplewas transferred into another tube and mixed with 100 μl of stop solution(400 mM NaCl and 100 mM EDTA) using mild agitation with a vortexer. Thesample was then mixed with 12 μl of 10% (w/v) sodium dodecyl sulfate(SDS) and incubated at 65° C. overnight. DNA was extracted with themixture of Tris-EDTA saturated phenol:chloroform:isoamyl alcohol(25:24:1, v/v). The extracted DNA was precipitated with 700 μl ofabsolute ethanol at 12,000 rpm for 30 minutes and washed with 70%ethanol. The DNA precipitate was air-dried and then dissolved in 10 μlTE buffer. The plasmid integrity was assessed by electrophoresis in a 1%agarose gel.

Formation of AWBP-PEG-g-PLL/pDNA complexes between negatively chargedplasmid DNA and positively charged AWBP-PEG-g-PLL was observed by gelband shift assay as shown in FIG. 3A. When a fixed amount of pCMV-Lucwas titrated with AWBP-PEG-g-PLL, the electrophoretic mobility of DNAwas retarded with increasing amount of AWBP-PEG-g-PLL. The complexes ofpDNA and AWBP-PEG-g-PLL in lanes 6-10 showed weaker band in fluorescenceintensity due to the exclusion of ethidium bromide after the formationof complexes. Complete complex formation was achieved at and above 1/1(+/−) charge ratio of DNA (FIG. 3A, lanes 6-10).

AWBP-PEG-g-PLL could protect pDNA from digestion with DNase for at least2 hours at 37° C. (FIG. 3B), whereas naked DNA was completely digestedby DNASE within 5 to 10 minutes of incubation at 37° C. (data notshown).

EXAMPLE 3

Particle size and morphology. The particle size of AWBP-PEG-g-PLL/pDNAcomplexes was measured by zeta potentiometer. AWBP-PEG-g-PLL/pDNAcomplexes were prepared as described above and diluted 4 times in thecuvette. The sample was subjected to mean particle size measurement byMalvern Zeta-Sizer 3000 (Malvern Instruments, U.K.) at 25° C., pH 7.0,and 677 nm wavelength with constant angle of 15°.

The morphology of AWBP-PEG-g-PLL/pDNA complexes was confirmed by atomicforce microscopy (AFM). Twenty microliters of AWBP-PEG-g-PLL/pDNAcomplexes (0.1 mg/ml) in PBS was placed on a MgAc₂ treated mica, A.Maheshwari et al., Soluble Biodegradable Polymer-based Cytokine GeneDelivery for Cancer Treatment, 2 Mol. Ther. 121-130 (2000), surface. Themica surface was rinsed gently with deionized water and dried withnitrogen gas. AFM images were obtained by Nanoscope II SFM (DigitalInstruments, Santa Barbara, Calif.) at room temperature with cantileveroscillation frequencies between 12 and 24 kHz.

The particle size of AWBP-PEG-g-PLL/pDNA was estimated as 85.9±5.3 nmwith relatively narrow and unimodal size distributions ranging from 70.8to 112.2 nm (FIG. 4) by zeta potentiometer. The morphology ofAWBP-PEG-g-PLL/pDNA complex was determined to be spherical shapes with adiameter around 100 nm by atomic force microscopy (AFM) (FIG. 5), thesedata were in agreement with the results from the zeta potentiometer.This suggests that AWBP-PEG-g-PLL/pDNA complexes possess an acceptablesize to enter the endosome of cells.

EXAMPLE 4

Gene expression. (Transfection assay) Primary bovine aorta endothelialcells and smooth muscle cells were prepared, cultured, characterized,and identified as described in L. Yu et al., supra. Bovine aortaendothelial cells (5×10⁵/well) and smooth muscle cells (2×10⁵/well) wereseeded in 24-well plates with 1 ml Dulbecco's modified Eagle medium(DMEM, Hyclone Laboratories, Logan, Utah) containing 10% fetal bovineserum (FBS, Hyclone Laboratories) and incubated for 24 hours to 70-80%confluency. The AWBP-PEG-g-PLL/pCMV-Luc complexes were freshly preparedin PBS for the transfection with fixed amount of plasmid DNA (2 μg/well)and various amounts of AWBP-PEG-g-PLL. After incubation of complexes for30 minutes at room temperature, 100 μl of complex solution was added tothe cells and then incubated for 3 hours at 37° C. in 5% CO₂ atmosphere.After replacement of media, the cells were incubated for 40 hours underthe same conditions. The cells were washed three times with PBS bufferand made ready for the reporter gene expression assay.

(Transfectioninhibition assay). Bovine aorta endothelial cells(5×10⁵/well) and smooth muscle cells (2×10⁵/well) were seeded in 24-wellplates 1 day prior to transfection with 70 to 80% confluence. TheAWBP-PEG-g-PLL/pCMV-Luc complexes were freshly prepared in PBS bufferfor the transfection with a fixed amount of plasmid DNA (2 μg/well) andAWBP-PEG-g-PLL (4 μg/well). After addition of various amounts of freeartery wall binding peptide (range from 31.3 μM to 1.0 mM) for 20minutes at 4° C., 100 μl AWBP-PEG-g-PLL/pCMV-Luc solution was added tothe cells. All the other conditions were the same as described above forthe transfection assay.

(Gene expression assay) Transgene expression was evaluated by luciferaseactivity of cell lysates from transfected bovine aorta endothelial cellsand smooth muscle cells. Measurement of luciferase activity wasperformed according to the manufacturer's instruction (Luciferase AssaySystem, Promega, Madison, Wis.). Briefly, the transfected cells werelysed with 1× lysis buffer (1% Triton X-100), 100 mM KPO₄, 2 mMdithiothreitol, 10% glycerol, and 2 mM EDTA, pH 7.8) for 15 minutes atroom temperature. To measure the luciferase activity, 20 μl aliquot ofcell lysate was mixed with 50 μl of luciferase assay reagent at roomtemperature and inserted in the luminometer. Light emission was measuredin triplicate over 10 s and expressed as relative light units (RLUs).RLUs were normalized from the protein content of each sample, which wasdetermined by BCA protein assay.

The transfection efficiencies of AWBP-PEG-g-PLL/pCMV-Luc complexes toartery wall cells were analyzed by in vitro transfection assay and invitro transfection inhibition assay. Luciferase activities of celllysate from both bovine aorta endothelial cells (FIG. 6A) and smoothmuscle cells (FIG. 6B) transfected with AWBP-PEG-g-PLL weresignificantly increased with the ratio of AWBP-PEG-g-PLL to plasmid DNAfrom 1.5:1 to 2:1, but remained constant with further increasing theratio from 2:1 to 5:1. This result indicated that AWBP-PEG-g-PLL/pDNAcomplexes were taken up by the artery wall cells underwent areceptor-mediated endocytosis pathway. The transfection efficiencies ofAWBP-PEG-g-PLL/pDNA complexes were 150-180 times higher than those ofcontrol systems such as PLL/pDNA and PEG-g-PLL/pDNA, regardless ofemployed charge ratios. These results indicate that incorporation ofAWBP to the PEG-g-PLL backbone was significantly enhanced the genetransfer to artery cell walls.

The luciferase activities of cell lysate from both bovine aortaendothelial cells (FIG. 7A) and smooth muscle cells (FIG. 7B)transfected with AWBP-PEG-g-PLL/pDNA complexes were significantlydecreased with an increase of free AWBP concentrations from 31.25 μM to500 μM. These results indicated that the existence of targeting moiety,free AWBP, could significantly inhibit gene transfer to artery cellwalls by AWBP-PEG-g-PLL/pDNA complexes. In the cases of control systemssuch as PLL/pDNA and PEG-g-PLL/pDNA, the luciferase activities were notfurther decreased with the increase of free AWBP concentration by 1000mM in both cell types (FIGS. 7A & 7B). These data demonstrated that genetransfection of AWBP-PEG-g-PLL/pDNA complexes to artery wall cellsproceeded via a specific receptor-mediated pathway related to AWBP.

1. A composition of matter comprising an artery wall binding peptide(SEQ ID NO:2) covalently coupled to a pharmaceutically acceptablecationic molecule, complex, or conjugate, wherein said cationicmolecule, complex, or conjugate is configured for complexing with anucleic acid.
 2. The composition of matter of claim 1 wherein saidartery wall binding peptide (SEQ ID NO:2) is present in a molar ratio tosaid cationic molecule, complex, or conjugate of greater than 1:1. 3.The composition of matter of claim 2 wherein said artery wall bindingpeptide (SEQ ID NO:2) is present in a molar ratio to said cationicmolecule, complex, or conjugate of at least 2:1.
 4. The composition ofmatter of claim 1 wherein said cationic molecule, complex, or conjugatecomprises a cationic polymer.
 5. The composition of matter of claim 4wherein said cationic polymer comprises poly(L-lysine).
 6. Thecomposition of matter of claim 4 wherein said cationic polymer comprisespoly(ethylene glycol)-grafted-poly(L-lysine).
 7. A composition of matterhaving the formula (AWBP)_(n)-PEG-g-PLL, wherein AWBP is an artery wallbinding peptide (SEQ ID NO: 2), n is an integer of at least 1, andPEG-g-PLL is poly(ethylene glycol)-grafted-poly(L-lysine).
 8. Thecomposition of matter of claim 7 wherein n is
 4. 9. A composition ofmatter comprising artery wall binding peptide (SEQ ID NO:2) covalentlycoupled to poly(ethylene glycol)-grafted-poly(L-lysine).
 10. Thecomposition of matter of claim 9 wherein artery wall binding peptide(SEQ ID NO:2) is covalently coupled to poly(ethyleneglycol)-grafted-poly(L-lysine) in a molar ratio of about 4:1.
 11. Amethod of making a composition having the formula: (AWBP)_(n)-PEG-g-PLL,wherein AWBP is an artery wall binding peptide (SEQ ID NO: 2), n is aninteger of at least 1, and PEG-g-PLL is poly(ethyleneglycol)-grafted-poly(L-lysine), comprising: (a) conjugatingpoly(ethylene glycol) to poly(L-lysine) to result in poly(ethyleneglycol)-grafted-poly(L-lysine); and (b) conjugating artery wall bindingpeptide (SEQ ID NO: 2) to the poly(ethyleneglycol)-grafted-poly(L-lysine) to result in (AWBP)_(n)-PEG-g-PLL. 12.The method of claim 11 wherein n is
 4. 13. A method for delivering anucleic acid to a cell in vitro bearing a receptor that binds an arterywall binding peptide (SEQ ID NO: 2) comprising: (a) mixing the nucleicacid with a composition of matter comprising an artery wall bindingpeptide (SEQ ID NO: 2) covalently coupled to poly(ethyleneglycol)-grafted-poly(L-lysine) to result in a complex comprising anucleic acid, a poly(ethylene glycol)-grafted-poly(L-lysine) and anartery wall binding peptide (SEQ ID NO: 2) and (b) causing the complexto contact the cell in vitro such that the receptor binds the arterywall binding peptide (SEQ ID NO: 2), thereby delivering the nucleic acidto the cell.
 14. The method of claim 13 wherein the artery wall bindingpeptide (SEQ ID NO:2) is covalently coupled to the poly(ethyleneglycol)-grafted-poly(L-lysine) in a molar ratio of about 4:1.